Antibody against antibacterial peptide and utiliazation thereof

ABSTRACT

An antibody which binds to a “peptide consisting of the amino acid sequence represented by SEQ ID NO:1”, and a method for assaying a “peptide comprising the amino acid sequence represented by SEQ ID NO:1” in a sample and a method for detecting a bacterial pneumonia, wherein the methods use the antibody or the like.

TECHNICAL FIELD

The present invention relates to an antibody which binds to a peptideconsisting of a specific amino acid sequence present in human-derivedCAP18 (cationic antimicrobial protein of 18 kDa; antibacterial protein),as well as an assaying method for. CAP18 and the like and an assayingkit which use the same.

BACKGROUND ART

JP-T-8-504085 (WO94/02589) discloses a full amino acid sequencecontaining a signal peptide of human-derived CAP18.

Minophagen Medical Review, vol. 43, No. 1, pp. 1-15 (1998) describes afull amino acid sequence of human-derived CAP18. Partial peptidesconsisting of 34, 32, 30, 27, 24 or 22 amino acid residues in theC-terminal region of human-derived CAP18 are also described.

Gendai-Iryo, vol. 28 (extra edition III), pp. 2367-2375 (1996) describesa full amino acid sequence of human-derived CAP18. Partial peptidesconsisting of 34, 30, 27, 24 or 22 amino acid residues in the C-terminalregion of human-derived CAP18 are also described.

Shock; From Molecular and Cellular Level to Whole Body (Proceedings ofthe Third International Shock Congress-Shock '95, Hamamatsu, Japan,21-23 October (1995)), and Okada, K., Ogata, H. eds. Elsevier SciencesB.V. pp. 109-115 (1996) describe a full amino acid sequence ofhuman-derived CAP18. Partial peptides consisting of 34, 30, 27 or 24amino acid residues in the C-terminal region of human-derived CAP18 isalso described. Data of these peptides regarding theirlipopolysaccharide (LPS) binding activity are also described.

Bacterial Endotoxins; Lipopolysaccharides From Genes to Therapy, Levin,J., Alving, C. R., Munford, R. S., Redl, H. eds. Wiley-Liss, Inc., NewYork, pp. 317-326 (1995) describes partial peptides consisting of 37 or32 amino acid residues in the C-terminal region of human-derived CAP18.

However, there is neither description nor suggestion about an antibodywhich binds to a partial peptide of CAP18 (peptide consisting of theamino acid sequence represented by SEQ ID NO:1). Moreover, there isneither description nor suggestion about a method for assaying a“peptide comprising the amino acid sequence represented by SEQ ID NO:1”such as CAP 8 using this antibody, a kit therefor, and the like.

If an antibody which binds to a peptide characteristic of CAP18 isobtained, it can be utilized as a tool for the detection and assay ofCAP18. Additionally, since the antibody is highly homogeneous andreproducible, and also can permanently be produced in a large amount,production costs can be remarkably reduced.

Also, if a method for assaying CAP18 or the like using the antibody oran assaying kit therefor is provided, it can be applied to a researchreagent or a diagnostic agent for diseases relating to CAP18 and thelike, and can also be possibly applied to monitoring of such diseases,etc.

DISCLOSURE OF THE INVENTION

The present invention provides an antibody which binds to a “peptideconsisting of the amino acid sequence represented by SEQ ID NO:1”(hereinafter referred to as “inventive antibody”).

The inventive antibody is preferably one which specifically binds to a“peptide consisting of the amino acid sequence represented by SEQ IDNO:2”, and this antibody is preferably a monoclonal antibody.Furthermore, preferably, the antibody belongs to the immunoglobulinsubclass IgG1.

Also, the inventive antibody is preferably one which specifically bindsto a “peptide consisting of the amino acid sequence represented by SEQID NO:3”, and the antibody is preferably a polyclonal antibody.

Also, the present invention provides a method for assaying a “peptidecomprising the amino acid sequence represented by SEQ ID NO:1” in asample, which comprises using an “inventive antibody” (hereinafterreferred to as “inventive method”).

The inventive method is preferably carried out by steps comprising thefollowing steps (a) and (b) (hereinafter referred to as “Inventivemethod-1”):

-   (a) bringing a solid phase into contact with a sample to thereby    immobilize the “peptide comprising the amino acid sequence    represented by SEQ ID NO:1” in the sample on the solid phase; and-   (b) detecting the “peptide comprising the amino acid sequence    represented by SEQ ID NO:1” immobilized on the solid phase in    step (a) by using an inventive antibody.

The “inventive antibody” used in Inventive method-1 is preferably onewhich is labeled with a label or is capable of being labeled with alabel. Furthermore, the detection of the “peptide comprising the aminoacid sequence represented by SEQ ID NO:1” is preferably carried out byfurther using an “antibody which specifically binds to the inventiveantibody and which is labeled with a label or is capable of beinglabeled with a label”.

Also, the inventive method is preferably carried out by steps comprisingthe following steps (a) and (b) (hereinafter referred to as “Inventivemethod-2”):

-   (a) bringing a “solid phase on which an inventive antibody (primary    antibody) is immobilized”, a “sample” and an “inventive antibody    (secondary antibody)” into contact to thereby form a sandwich    complex of “the primary antibody immobilized on the solid phase—the    peptide comprising the amino acid sequence represented by SEQ ID    NO:1—the secondary antibody”; and-   (b) detecting the sandwich complex formed in step (a).

Also, the method is more preferably carried out by steps comprising thefollowing steps (a) to (c):

-   (a) bringing a sample into contact with a “solid phase on which an    inventive antibody (primary antibody) is immobilized” to thereby    form a complex of “the primary antibody immobilized on the solid    phase—the peptide comprising the amino acid sequence represented by    SEQ ID NO:1”;-   (b) bringing an “inventive antibody (secondary antibody)” into    contact with the solid phase to thereby form a sandwich complex of    “the primary antibody immobilized on the solid phase—the peptide    comprising the amino acid sequence represented by SEQ ID NO:1—the    secondary antibody”; and-   (c) detecting the sandwich complex formed in step (b).

The “secondary antibody” used in Inventive method-2 is preferably onewhich is labeled with a label or is capable of being labeled with alabel. Furthermore, the detection of the complex in Inventive method-2is preferably carried out by using an “antibody which specifically bindsto the secondary antibody and which is labeled with a label or iscapable of being labeled with a label”.

Also, the inventive method is preferably carried out by steps comprisingthe following steps (a) and (b) (hereinafter referred to as “Inventivemethod-3”):

-   (a) bringing a “solid phase on which a peptide consisting of the    amino acid sequence represented by SEQ ID NO:1 is immobilized”, a    “sample” and an “inventive antibody” into contact to thereby form a    complex of “the peptide consisting of the amino acid sequence    represented by SEQ ID NO:1 immobilized on the solid phase—the    inventive antibody” and a complex of “the peptide comprising the    amino acid sequence represented by SEQ ID NO:1 in the sample—the    inventive antibody”; and-   (b) detecting at least one of the complex of “the peptide consisting    of the amino acid sequence represented by SEQ ID NO:1 immobilized on    the solid phase—the inventive antibody” and the complex of “the    peptide comprising the amino acid sequence represented by SEQ ID    NO:1 in the sample—the inventive antibody”.

This method is more preferably carried out by steps comprising thefollowing steps (a) to (c):

-   (a) bringing a sample into contact with an “inventive antibody” to    thereby form a complex-A of “the peptide comprising the amino acid    sequence represented by SEQ ID NO:1—the inventive antibody”;-   (b) bringing a “mixture comprising the complex-A and the inventive    antibody which does not form the complex-A” obtained in step (a)    into contact with a “solid phase on which a peptide consisting of    the amino acid sequence represented by SEQ ID NO:1 is immobilized”    to thereby form a complex of “the peptide consisting of the amino    acid sequence represented by SEQ ID NO:1 immobilized on the solid    phase—the inventive antibody”; and-   (c) detecting the complex formed in step (b).

The “inventive antibody” used in Inventive method-3 is preferably onewhich is labeled with a label or is capable of being labeled with alabel. It is also preferable to carry out the detection of the complexin Inventive method-3 by using an “antibody which specifically binds tothe inventive antibody and which is labeled with a label or is capableof being labeled with a label”.

The “sample” used in the inventive methods is preferably a body fluid.

Also, the present invention provides a kit for assaying a “peptidecomprising the amino acid sequence represented by SEQ ID NO:1”, whichcomprises the following components (A) and (B) (hereinafter referred toas “Inventive kit-1”):

-   (A) a solid phase; and-   (B) an inventive antibody.

The “inventive antibody” used in Inventive kit-1 is preferably one whichis labeled with a label or is capable of being labeled with a label.

Also, the present invention provides a kit for assaying a “peptidecomprising the amino acid sequence represented by SEQ ID NO:1”, whichcomprises the following components (A) and (B) (hereinafter referred toas “Inventive kit-2”):

-   (A) a solid phase on which an inventive antibody (primary antibody)    is immobilized; and-   (B) an inventive antibody (secondary antibody).

The “inventive antibody” used in Inventive kit-2 is preferably one whichis labeled with a label or is capable of being labeled with a label.

Also, the present invention provides a kit for assaying a “peptidecomprising the amino acid sequence represented by SEQ ID NO:1”, whichcomprises the following components (A), (B) and (C) (hereinafterreferred to as “Inventive kit-3”):

-   (A) a solid phase on which a peptide consisting of the amino acid    sequence represented by SEQ ID NO:1 is immobilized;-   (B) an inventive antibody; and-   (C) an antibody which specifically binds to the inventive antibody    and which is labeled with a label or is capable of being labeled    with a label.

Also, the present invention provides a method for detecting a bacterialpneumonia, which comprises assaying an antigen in a sample which can bedetected by an “inventive antibody” or an “antibody capable ofspecifically binding to CAP18” to thereby detect a bacterial pneumoniain a patient from which the sample is obtained (hereinafter referred toas “inventive detection method”).

In the inventive detection method, the antigen in the sample ispreferably an antigen selected from the group consisting of a “peptidecomprising the amino acid sequence represented by SEQ ID NO:1”, a“peptide comprising the amino acid sequence represented by SEQ ID NO:2”,a “peptide comprising the amino acid sequence represented by SEQ IDNO:3” and CAP18.

In the inventive detection method, preferably, the assay isimmunologically carried out by using an antibody selected from the groupconsisting of an “antibody capable of binding to a peptide consisting ofthe amino acid sequence represented by SEQ ID NO:1”, an “antibodycapable of specifically binding to a peptide consisting of the aminoacid sequence represented by SEQ ID NO:2”, an “antibody capable ofspecifically binding to a peptide consisting of the amino acid sequencerepresented by SEQ ID NO:3” and an “antibody capable of specificallybinding to CAP18”.

In the inventive detection method, the detection of a bacterialpneumonia is preferably carried out by evaluating or monitoring thepresence or absence of infection, degree or type of the bacterialpneumonia.

Also, in the inventive detection method, the assay is preferably carriedout by the above inventive method.

The present invention provides a kit for diagnosing a bacterialpneumonia, which comprises an “inventive antibody” (hereinafter referredto as “inventive diagnostic kit”).

The inventive diagnostic kit is preferably a kit consisting of any oneof Inventive kits 1 to 3.

The present invention provides a diagnostic agent which comprises, as anactive ingredient, an “inventive antibody” (hereinafter referred to as“inventive diagnostic agent”).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is photographs showing immunological staining images of cellsusing a monoclonal antibody Toyo6E3 (morphology of an organism).

FIG. 2 shows detection and determination results of a peptide by directELISA.

FIG. 3 shows detection and determination results of CAP18 by indirectELISA (sandwich method).

FIG. 4 is a photograph showing the results of a detection of CAP18 incell extracts by Western blotting.

FIG. 5 is a photograph showing detection results of serum CAP18 byimmunoprecipitation.

FIG. 6 shows schematic views (A and B) and a photograph (C) of detectionand determination results of release of CAP18 from human neutrophilesstimulated with fMLP.

BEST MODE FOR CARRYING OUT THE INVENTION

The present inventors made an effort to solve the above problems andcould provide an antibody which binds to a peptide consisting of theamino acid sequence represented by SEQ ID NO:1. Also, the presentinventors could provide a method for assaying a “peptide comprising theamino acid sequence represented by SEQ ID NO:1” such as CAP18 and anassaying kit therefor, which use the antibody. Thus, the presentinvention has been completed.

Embodiments of the present invention are described below.

<1> Inventive Antibody

A full amino acid sequence comprising a human-derived CAP18 signalpeptide part is represented by SEQ ID NO:4. The signal peptide partcorresponds to the −30th to −1st amino acids in SEQ ID NO:4.

The inventive antibody is an antibody which binds to a “peptideconsisting of the amino acid sequence represented by SEQ ID NO:1”. Theamino acid sequence represented by SEQ ID NO:1 corresponds to the 109thto 135th amino acids in SEQ ID NO:4.

The inventive antibody can be obtained by the usual antibody productionmethod using, as an antigen, a peptide consisting of the amino acidsequence represented by SEQ ID NO:1 (peptide of SEQ ID NO:1 itself) or apartial peptide thereof (hereinafter sometimes referred to as “antigenpeptide”).

The partial peptide includes a peptide consisting of the amino acidsequence represented by SEQ ID NO:2 and a peptide consisting of theamino acid sequence represented by SEQ ID NO:3. The amino acid sequencerepresented by SEQ ID NO:2 corresponds to the 118th to 135th amino acidsin SEQ ID NO:4, and the amino acid sequence represented by SEQ ID NO:3corresponds to the 109th to 117th amino acids in SEQ ID NO:4.

The antigen peptide can be produced based on its sequence by a knownmethod for chemically synthesizing a peptide (for example, a liquidphase synthesis, solid phase synthesis, etc.; see Nobuo Izumiya, TetsuoKato, Tohiko Aoyagi, Michinori Waki, Fundamentals and Experiments ofPeptide Syntheses, 1985, Maruzen).

Also, the antigen peptide can be produced by producing a polynucleotide(DNA or RNA) corresponding to the amino acid sequence of the antigenpeptide and applying gene engineering techniques using thepolynucleotide.

In this connection, a “peptide comprising the amino acid sequencerepresented by SEQ ID NO:1” such as CAP18 can be used as an antigenpeptide, so long as an antibody which binds to a “peptide consisting ofthe amino acid sequence represented by SEQ ID NO:1” can be obtained. Insuch a case, since an antibody which binds to a peptide other than the“peptide consisting of the amino acid sequence represented by SEQ IDNO:1” may also be obtained, a “peptide consisting of the amino acidsequence represented by SEQ ID NO:1” is used to select an antibodybinding thereto.

An antigen peptide thus produced can be purified by a method forisolating and purifying a peptide which is well known in the art of theprotein chemistry. Examples include extraction, recrystallization,salting out using ammonium sulfate, sodium sulfate, etc.,centrifugation, dialysis, ultrafiltration, absorption chromatography,ion exchange chromatography, hydrophobic chromatography, normal-phasechromatography, reverse-phase chromatography, gel filtration, gelpermeation chromatography, affinity chromatography, electrophoresis,countercurrent distribution, and the like, and combinations thereof.

The amino acid sequence of the antigen peptide produced is determined bya known amino acid sequencing method (for example, Edman degradationmethod, etc.) to confirm that the antigen peptide has been correctlyproduced.

When a relatively low molecular peptide such as a peptide consisting ofan amino acid sequence of SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3 isused as an antigen, the antigen is preferably bound to a carrier such ashemocyanin, ovalbumin or γ-globulin.

The inventive antibody may be a monoclonal antibody or a polyclonalantibody. The production of the inventive antibody can be accomplishedas follows depending on the type of the antibody desired, i.e., amonoclonal antibody or a polyclonal antibody.

A monoclonal inventive antibody can be produced by using an antigenpeptide described above by a method of Kohler and Milstein (Nature, 256,495-497 (1975)).

For example, an antigen peptide is administered intraperitoneally,subcutaneously or to a footpad of an animal to be immunized such as amouse, a rat, a guinea pig, a rabbit, a goat, a sheep, a horse, a pig, adog, a cat or a chicken. Among these animals to be immunized, a mouse ispreferably used. Thus, the inventive antibody is preferably amouse-derived antibody.

From an animal to be immunized, a spleen cell, a lymphocyte, peripheralblood or the like is collected and is subjected to cell fusion with amyeloma cell which is a tumor cell line to prepare a hybridoma. Althoughthe myeloma cell used in the cell fusion may be a cell line from any ofvarious mammalian animals, it is preferred to use a cell line of ananimal homologous to the immunized animal. Furthermore, in order todistinguish the non-fused cell and the fused cell, the myeloma cellhaving a marker is preferably used so as to proliferate only a hybridomawithout survival of the non-fused myeloma cell. Moreover, since anantibody of interest is easily obtained from the culture supernatant ofthe hybridoma, a myeloma cell line which does not secret a specificimmunoglobulin is preferably used.

The resulting hybridoma is continuously proliferated, and a hybridomacell line which continuously produces an antibody capable ofspecifically binding to an antigen is selected.

The hybridoma cell line thus selected is cultured in a suitable mediumto obtain a monoclonal antibody in the culture. It is also possible toproduce a monoclonal antibody in a large amount by culturing thehybridoma cell line described above in vivo, for example, in theabdominal cavity in a mouse, followed by isolation from the ascites. Themonoclonal antibody thus obtained may be purified by the usual antibodypurification method.

A polyclonal inventive antibody can be produced as described below byusing the above-described antigen peptide.

Similarly to the above-described production method of the monoclonalantibody, an antigen peptide is administered to an animal to beimmunized. Herein, a rabbit is preferably used as the animal to beimmunized.

When the animal to be immunized is immunized, an adjuvant is preferablyused in combination since an antibody-producing cell is activated. Whenbooster immunization is carried out 2 to 3 weeks after the firstimmunization serves by the usual method, antiserum having a high titercan be produced. About 1 week after the final immunization, blood iscollected and serum is separated. The serum is heated to inactivate acomplement, and then an immunoglobulin fraction can be purified by theusual antibody purification method.

The antibody purification method includes salting out using sodiumsulfate, ammonium sulfate, etc., low temperature alcohol precipitation,selective precipitation separation using polyethylene glycol or anisoelectric point, electrophoresis, ion exchange chromatography by usingan ion exchanger such as DEAE (diethylaminoethyl)-derivative or CM(carboxymethyl)-derivative, affinity chromatography using protein A orprotein G, hydroxyapatite chromatography, antigen-immobilizingimmunosorbent chromatography, gel filtration, supercentrifugation, andthe like.

The inventive antibody can be treated with a protease which does notdecompose an antigen binding site (Fab) (for example, plasmin, pepsin,papain, etc.) to form an Fab-containing fragment. The Fab-containingfragment of the antigen includes Fabc, (Fab′)₂ and the like, in additionto Fab. They are included in the concept of the “inventive antibody” inthe present specification.

Also, when the nucleotide sequence of a gene encoding the inventiveantibody or the amino acid sequence of the inventive antibody isdetermined, a fragment containing Fab of the inventive antibody or achimera antibody (for example, a chimera antibody containing Fab of theinventive antibody, etc.) by genetic engineering techniques. Thefragment or chimera antibody containing Fab of the inventive antibodymay also be included in the concept of the “inventive antibody” in thepresent specification, so long as it binds to an antigen peptide.

Whether or not the produced antibody binds to the antigen peptide, orwhether or not the antibody specifically binds to the antigen peptidecan easily be determined by those skilled in the art by the usual methodusing other substance which can be used as an antigen peptide or anantigen (for example, peptide of other kind) or the like.

Preferably, the inventive antibody belongs to the immunoglobulinsubclass IgG1. The antibody belonging to the immunoglobulin subclassIgG1 can be obtained, for example, by a screening method using ananti-IgG1 antibody or the like.

Also, other preferred embodiment of the inventive antibody is anantibody which specifically binds to a “peptide consisting of the aminoacid sequence represented by SEQ ID NO:3”. This antibody is preferably apolyclonal antibody.

The inventive antibody may be one which is labeled with a label or iscapable of being labeled with a label. Although the label which can beused for labeling is not particularly limited, so long as it canordinarily be used for labeling a protein. Examples include an enzyme(e.g., peroxidase, alkaline phosphatase, β-galactosidase, luciferase,acetylcholine esterase, glucose oxidase, etc.), a radioisotope (¹²⁵I,¹³¹I, ³H, etc.), a fluorescent dye (Alexa Fluor® 488, fluoresceinisothiocyanate (FITC), 7-amino-4-methylcoumarine-3-acetic acid (AMCA),dichlorotriazinyl aminofluorescein (DTAF), tetramethyl Rhodamineisothiocyanate (TRITC), Lussamine rhodamine B, Texas red, phycoerythrin(PE), umbeliferon, europium, phycocyanin, Tricolor, cyanin, etc.), achemiluminescent substance (luminol, etc.), hapten(dinitrofluorobenzene, adenosine monophosphate (AMP),2,4-dinitroaniline, etc.), either member of a specific binding pair(biotin and avidins (streptoavidin, etc.), lectin and a saccharidechain, an agonist and an agonist receptor, heparin and antithrombin III(ATIII), a polysaccharide and its binding protein (hyaluronic acid and ahyaluronic acid-binding protein (HABP), etc.)), and the like.

A method for labeling the inventive antibody with a label can beappropriately selected from known methods suitable for the label, forexample, in the case of labeling the inventive antibody with an enzyme,from a glutaraldehyde method, a periodic acid crosslinking method, amaleimide crosslinking method, a carbodiimide method, an activated estermethod, and the like, and in the case of labeling the inventive antibodywith a radioisotpoe, from a chloramine T method, a lactoperoxidasemethod, and the like (see, Sequel of Biochemistry Experiments, 2,“Protein Chemistry (2nd vol.)”, Tokyo Kagaku Dojin (1987)). For example,in the case of using biotin as a label, a method using anN-hydroxysuccinimide ester derivative of biotin or a hydrazidederivative can be used (see, Avidin-Biotin Chemistry: A Handbook, p.57-63, Pierce Chemical Company, 1994).

Also, when the inventive antibody is stored, distributed or used, othercomponents can be added, so long as the functions and effects of theinventive antibody are not substantially damaged. For example, it cancontain an excipient, a buffering agent, a stabilizer, a preservativeand the like which are used in preparing a usual reagent. Examplesinclude phosphate buffered saline (PBS), sodium azide (NaN₃), bovineserum albumin (BSA), and the like.

<2> Inventive Method

The inventive method is a method for assaying a “peptide comprising theamino acid sequence represented by SEQ ID NO:1” in a sample, whichcomprises using an “inventive antibody”.

The explanation of the “inventive antibody” used in the inventive methodis similar to that of the “inventive antibody” described above. Theinventive antibody is preferably one which is labeled with a label or iscapable of being labeled with a label in order to facilitate thedetection, The label used for labeling and the labeled method aresimilar to those described above.

Also, the “sample” is not particularly limited, and includes onecontaining a “peptide comprising the amino acid sequence represented bySEQ ID NO:1” such as CAP18 and one which is capable of containing it.Examples include a standard solution of CAP18, a cell or tissue extract,a cell culture supernatant, a body fluid and the like, and a body fluidis preferred. The “body fluid” includes, for example, blood (used as aconcept including serum and plasma in the present specification), urine,saliva, sweat, tear, an intraarticular fluid, and the like.

Also, the “peptide comprising the amino acid sequence represented by SEQID NO:1” which is assayed is not particularly limited, so long as itcontains at least the amino acid sequence represented by SEQ ID NO:1,and it is a concept including polypeptides. Examples include CAP18, apartial peptide thereof (comprising the amino acid sequence representedby SEQ ID NO:1), a peptide itself consisting of the amino acid sequencerepresented by SEQ ID NO:1, and the like.

A method for “assaying” a “peptide comprising the amino acid sequencerepresented by SEQ ID NO:1” in a sample is not particularly limited, solong as it is a method capable of detecting the peptide by using theinventive antibody. In this connection, the “assaying” in the inventivemethod is a concept including not only a quantitative detection but alsoa qualitative detection (detection of the presence or absence).Accordingly, the “assaying” in the present invention includes screeningof the peptide in a sample.

A method for assaying a “peptide comprising the amino acid sequencerepresented by SEQ ID NO:1” by using the inventive antibody includesthose described below.

-   i) A method in which a “peptide comprising the amino acid sequence    represented by SEQ ID NO:1” in a sample is immobilized on a solid    phase and detected directly (so-called direct ELISA).-   ii) A method in which a sample is brought into contact with a solid    on which an inventive antibody (primary antibody) is immobilized and    then with an inventive antibody (secondary antibody) to thereby form    a sandwich complex, and then the complex is detected (so-called    sandwich method).-   iii) A method in which a “peptide consisting of the amino acid    sequence represented by SEQ ID NO:1” immobilized on a solid phase, a    sample and an inventive antibody (the sample and the inventive    antibody may be brought into contact in advance) are allowed to    coexist while allowing the “peptide consisting of the amino acid    sequence represented by SEQ ID NO:1” immobilized on the solid phase    and a “peptide comprising the amino acid sequence represented by SEQ    ID NO:1” in the sample to compete with each other, and the inventive    antibody bound to the solid phase is detected to thereby assay the    “peptide comprising the amino acid sequence represented by SEQ ID    NO:1” in the sample (so-called inhibition method).-   iv) A method in which a sample is brought into contact with    microparticles on which an inventive antibody is immobilized and    then with an inventive antibody to thereby aggregate the    microparticles, and then the aggregate (or precipitate) is detected    (so-called aggregation method).    <2>-1 Inventive Method-1

The inventive method is preferably carried out by direct ELISA.Specifically, the inventive method is preferably carried out by stepscomprising the following steps (a) and (b) (Inventive method-1):

-   (a) bringing a sample into contact with a solid phase to thereby    immobilize a “peptide comprising the amino acid sequence represented    by SEQ ID NO:1” in the sample on the solid phase;-   (b) detecting the “peptide comprising the amino acid sequence    represented by SEQ ID NO:1” immobilized on the solid phase in    step (a) by using the inventive antibody.

Each step is discussed in detail below.

Step (a):

Step (a) is a step for bringing a solid phase into contact with a sampleto thereby immobilize the “peptide comprising the amino acid sequencerepresented by SEQ ID NO:1” in the sample on the solid phase. The “solidphase” on which the “peptide comprising the amino acid sequencerepresented by SEQ ID NO:1” in the sample is immobilized is notparticularly limited, so long as it is capable of immobilizing thepeptide and is insoluble in water, a sample or an assaying reactionsolution. The shape of the solid phase includes a plate (e.g., well ofmicroplate, etc.), a tube, a bead, membrane, gel, a microparticulatesolid carrier (gelatin particle, kaolin particle, synthetic polymerparticle such as latex, etc.) and the like. Among these, a microplate ispreferred in view of accurate determination and convenient handling.

The material of the solid phase includes polystyrene, polypropylene,polyvinyl chloride, nitrocellulose, nylon, polyacrylamide, Teflon®,polyaromer, polyethylene, glass, agarose, and the like. Among these, aplate made of polystyrene is preferred.

As the method for immobilizing the “peptide comprising the amino acidsequence represented by SEQ ID NO:1” in the sample to the solid phase, ausual method for preparing an immobilized enzyme such as a physicaladsorption method, a covalent bond method and an inclusion method can beused (see, Immobilized Enzymes, 1975, Kodansha, pp. 9 to 75).

Among these, a physical adsorption method is preferred in view of itssimple operation and wide use.

A specific method for physical adsorption is described below.

A sample is diluted in a buffer at pH 7 to 10 (for example, carbonatebuffer, phosphate buffer, PBS, etc.) and added to a solid phase (forexample, microplate), followed by storing at about 20 to 37° C. for 1 to2 hours or at 4° C. overnight for immobilization.

The explanations of the “sample”” used herein and the “peptidecomprising the amino acid sequence represented by SEQ ID NO:1” to beassayed are similar to those described above.

The method for bringing a solid phase into contact with a sample is notparticularly limited, so long as the solid phase is brought into contactwith the “peptide comprising the amino acid sequence represented by SEQID NO:1” in the sample. For example, the contact can be carried out byadding the sample to the solid phase, the contact can be carried out byadding the solid phase to the sample, or both can be addedsimultaneously to a separate container. The method of the contact is notlimited thereto, and may be appropriately determined by those skilled inthe art based on the shape and material of the solid phase, and thelike.

After bringing the solid phase into contact with the sample, the solidphase and the liquid phase are separated. It is preferred to wash thesurface of the solid phase with a washing solution, if necessary, toremove any non-specifically adsorbed substances or non-reactedcomponents in the sample.

The washing solution is preferably a buffer (for example, phosphatebuffer, PBS, Tris-HCl buffer, etc.) to which a nonionic surfactant suchas a Tween-based surfactant is added.

The solid phase is brought into contact with a sample to therebyimmobilize the “peptide comprising the amino acid sequence representedby SEQ ID NO:1” in the sample on the solid phase.

Step (b):

Step (b) is a step for detecting the “peptide comprising the amino acidsequence represented by SEQ ID NO:1” immobilized on the solid phase instep (a) by using the inventive antibody. The explanation of theinventive antibody is similar to that described above.

The method for detecting the “peptide comprising the amino acid sequencerepresented by SEQ ID NO:1” immobilized on the solid phase is notparticularly limited, so long as the detection is carried out by usingthe inventive antibody. For example, when the inventive antibody islabeled with a label, the inventive antibody is bound to the “peptidecomprising the amino acid sequence represented by SEQ ID NO:1”immobilized on the solid phase, and the label is then detected tothereby detect the peptide.

The method for detecting the label may be appropriately selected fromknown detection means depending on the type of the label. For example,in the case where one member of a specific binding pair (for example,biotin) is used as the label, an enzyme (for example, peroxidase, etc.)bound to the other member which specifically binds thereto (for example,streptoavidin) is added to thereby form a specific binding pair. Then, asubstrate for the enzyme (for example, hydrogen peroxide (in the casewhere the enzyme is peroxidase)) and a chromogenic substance (forexample, 3,3′,5,5′-tetramethylbenzidine (TMB), diaminobenzidine, etc.)are added and the degree of the color development by a product of theenzyme reaction is assayed based on an absorbance to thereby detect thelabel.

Also, when a radioisotope, a fluorescent dye or a chemiluminescentsubstance is used as a label, the method includes methods assaying aradioactivity count, fluorescent intensity, fluorescent polarization,luminescent intensity and the like.

By such detection of the label, the “peptide comprising the amino acidsequence represented by SEQ ID NO:1” immobilized on the solid phase canbe detected to thereby assay the “peptide comprising the amino acidsequence represented by SEQ ID NO:1” in the sample. Since this method isdirect ELISA, detection of the label in a large amount means that the“peptide comprising the amino acid sequence represented by SEQ ID NO:1”immobilized on the solid phase is present in a large amount, i.e., the“peptide comprising the amino acid sequence represented by SEQ ID NO:1”is present in a large amount in the sample.

When a qualitative assay (detection of the presence or absence) of the“peptide comprising the amino acid sequence represented by SEQ ID NO:1”is desired, the presence or absence of the detection of the label can beused, as it is, as an assaying result.

Alternatively, when a quantitative assay (assay of the concentration,etc.) of the “peptide comprising the amino acid sequence represented bySEQ ID NO:1” is desired, the absorbance, radioactivity count,fluorescent intensity, luminescent intensity and the like can be used,as they are, as an index of the quantity of the “peptide comprising theamino acid sequence represented by SEQ ID NO:1”. Furthermore, acalibration curve or equation for the relationship between the “peptidecomprising the amino acid sequence represented by SEQ ID NO:1” and thedetection results (for example, absorbance) of the standard substance isprepared in advance by using a standard solution of the “peptidecomprising the amino acid sequence represented by SEQ ID NO:1” having aknown concentration, and the concentration of the “peptide comprisingthe amino acid sequence represented by SEQ ID NO:1” in the sample can beobtained based on it. Also, when urine is used as a sample, theresultant concentration can be corrected on the basis of theconcentration of other component in the urine, such as creatinine.

Moreover, the detection of the “peptide comprising the amino acidsequence represented by SEQ ID NO:1” immobilized on the solid phase ispreferably carried out by using an “antibody which specifically binds tothe inventive antibody and which is labeled with a label or is capableof being labeled with a label”.

The “antibody which specifically binds to the inventive antibody” is notparticularly limited, so long as it specifically binds to the inventiveantibody. For example, depending on an animal from which the inventiveantibody is derived or an immunoglobulin class, an antibody whichspecifically binds to the immunoglobulin class of the animal isexemplified. For example, when the inventive antibody is animmunoglobulin (mouse-derived IgG1), the anti-mouse IgG1 antibody can beused as an “antibody which specifically binds to the inventiveantibody”. The label which can be used for labeling the “antibody whichspecifically binds to the inventive antibody”, the labeling method, thelabel detecting method and the like are similar to those described inthe above.

<2>-2 Inventive Method-2

The inventive method is also preferably carried out by a sandwichmethod. Specifically, the inventive method is also preferably carriedout by steps comprising the following steps (a) and (b) (Inventivemethod-2):

-   (a) bringing a “solid phase on which an inventive antibody (primary    antibody) is immobilized”, a “sample” and an “inventive antibody    (secondary antibody)” into contact to thereby form a sandwich    complex of “the primary antibody immobilized on the solid phase—the    peptide comprising the amino acid sequence represented by SEQ ID    NO:1—the secondary antibody”;-   (b) detecting the sandwich complex formed in step (a).

In step (a), the three members, i.e., the “solid phase on which aninventive antibody (primary antibody) is immobilized”, the “sample” andthe “inventive antibody (secondary antibody)” can be brought intocontact simultaneously, or, alternatively, the former two are firstbrought into contact and then into contact with the latter one, or thelatter two are first brought into contact and then into contact with theformer one. Among these, preferably, the former two are first broughtinto contact and then into contact with the latter one. That is, themethod is more preferably carried out by steps comprising the followingsteps (a) to (c):

-   (a) bringing a sample into contact with a “solid phase on which an    inventive antibody (primary antibody) is immobilized” to thereby    form a complex of “the primary antibody immobilized on the solid    phase—the peptide comprising the amino acid sequence represented by    SEQ ID NO:1”;-   (b) bringing an “inventive antibody (secondary antibody)” into    contact with the solid phase described above to thereby form a    sandwich complex of “the primary antibody immobilized on the solid    phase—the peptide comprising the amino acid sequence represented by    SEQ ID NO:1—the secondary antibody”; and-   (c) detecting the sandwich complex formed in step (b).

Each step is discussed in detail below.

Step (a):

Step (a) is a step for bringing a sample into contact with a “solidphase on which an inventive antibody (primary antibody) is immobilized”to thereby form a complex of “the primary antibody immobilized on thesolid phase—the peptide comprising the amino acid sequence representedby SEQ ID NO:1”.

The explanation of the “inventive antibody (primary antibody)” issimilar to that described above.

Also, the explanation of the “solid phase” on which the inventiveantibody (primary antibody) is immobilized is similar to that inInventive method-1. Furthermore, the method for immobilizing theinventive antibody (primary antibody) on the solid phase is similar tothat in Inventive method-1, and a usual method such as a physicaladsorption method, a covalent bond method or an inclusion method can beused. Among these, a physical adsorption method is preferred because ofits simple operation and wide use.

A specific method for physical adsorption is described below.

An inventive antibody (primary antibody) is diluted in a buffer at pH 7to 9 (for example, Tris buffer, phosphate buffer, PBS, carbonate buffer,etc.) and added to a solid phase (for example, microplate), followed bystoring at about 20 to 37° C. for 1 to 2 hours or at 4° C. overnight tothereby immobilize the inventive antibody (primary antibody) on thesolid phase.

Also, a part of the surface on which the inventive antibody (primaryantibody) is not immobilized may remain on the surface of the solidphase on which the inventive antibody (primary antibody) is immobilized.When the “peptide comprising the amino acid sequence represented by SEQID NO:1” present in the sample is non-specifically immobilized thereon,correct assaying results might not be obtained. Accordingly, it ispreferred that prior to the contact of the sample with the solid phase,a blocking agent is added to cover the part where the inventive antibody(primary antibody) is not immobilized. The blocking agent includesserum, serum albumin, casein, skimmed milk, gelatin, pluronic and thelike, and those which are commercially available as blocking agents canbe used.

Specifically, the method for blocking includes a method in which ablocking agent is added, followed by storing at about 37° C. for 30minutes to 2 hour or at room temperature (15 to 25° C.) for 1 to 2hours.

The explanation of the “sample” used herein is similar to that describedabove.

The method for bringing the solid phase into contact with a sample isnot particularly limited, so long as the inventive antibody (primaryantibody) immobilized on the solid phase is brought into contact withthe “peptide comprising the amino acid sequence represented by SEQ IDNO:1” in the sample. Other explanations of the “method for bringing asolid phase into contact with a sample” are similar to those inInventive method-1.

After both are brought into contact, the reaction is preferably carriedout at 4 to 37° C., preferably 20 to 37° C., for about 1 to 4 hours inorder to sufficiently bind the inventive antibody (primary antibody) andthe “peptide comprising the amino acid sequence represented by SEQ IDNO:1” in the sample.

After the reaction, the solid phase and the liquid phase are separated.It is preferred to wash the surface of the solid phase with a washingsolution, if necessary, in order to remove any non-specifically adsorbedsubstances or non-reacted components in the sample. The explanation ofthe washing solution used herein is similar to that in Inventivemethod-1.

A complex of “the inventive antibody immobilized on the solid phase(primary antibody)—the peptide comprising the amino acid sequencerepresented by SEQ ID NO:1” is formed by bringing the sample intocontact with the solid phase on which the inventive antibody (primaryantibody) is immobilized.

Step (b):

Step (b) is a step for bringing an “inventive antibody (secondaryantibody)” into contact with the solid phase after step (a) to therebyform a sandwich complex of “the primary antibody immobilized on thesolid phase—the peptide comprising the amino acid sequence representedby SEQ ID NO:1—the secondary antibody”.

The explanation of the “inventive antibody (secondary antibody)” issimilar to that described above.

The secondary antibody is preferably labeled with a label or is capableof being labeled with a label in order to facilitate the detection. Thelabel used in labeling and the method for labeling are similar to thosedescribed above.

The contact between the solid phase described above after step (a) andthe inventive antibody (secondary antibody) can be carried out similarlyto the “method for bringing a solid phase into contact with a sample”described above. Similarly to the above, the solid phase and the liquidphase are separated after the reaction and it is preferred that thesurface of the solid phase is washed with a washing solution, ifnecessary, in order to remove any non-specifically adsorbed substancesor non-reacted components in the sample. The washing solution which canbe used is also similar to that described above.

The secondary antibody is brought into contact with the above-describedsolid phase after step (a) (wherein a complex of “the primary antibodyimmobilized on the solid phase—the peptide comprising the amino acidsequence represented by SEQ ID NO:1” is formed) to thereby form asandwich complex of “the primary antibody immobilized on the solidphase—the peptide comprising the amino acid sequence represented by SEQID NO:1—the secondary antibody” is formed.

Step (c):

Step (c) is a step for detecting the sandwich complex formed in step(b).

The method for detecting the sandwich complex is not particularlylimited. For example, when the secondary antibody is labeled with alabel, the label is detected to thereby detect the complex.

The method for detecting the label may be appropriately selected fromknown detection means depending on the type of the label. Otherexplanations are similar to those in Inventive method-1.

By the detection of the label, the sandwich complex can be detected tothereby assay the “peptide comprising the amino acid sequencerepresented by SEQ ID NO:1” in the sample. Since this method is asandwich method, detection of the label in a large amount means that thesandwich complex is present in a large amount, i.e., the “peptidecomprising the amino acid sequence represented by SEQ ID NO:1” ispresent in a large amount in the sample.

The explanations of a qualitative assay (detection of the presence orabsence) and a quantitative assay (e.g., assay of the concentration,etc.) of the “peptide comprising the amino acid sequence represented bySEQ ID NO:1” are similar to those in Inventive method-1.

Furthermore, the detection of the complex in Inventive method-2 ispreferably carried out by using an “antibody which specifically binds tothe secondary antibody and which is labeled with a label or is capableof being labeled with a label”.

The explanation of the “antibody which specifically binds to thesecondary antibody (inventive antibody)” is also similar to that of the“antibody which specifically binds to the inventive antibody” inInventive method-1.

<2>-3 Inventive Method-3

The inventive method is also preferably carried out by an inhibitionmethod. Specifically, the inventive method is also preferably carriedout by steps comprising the following steps (a) and (b) (Inventivemethod-3):

-   (a) bringing a “solid phase on which a peptide consisting of the    amino acid sequence represented by SEQ ID NO:1 is immobilized”, a    “sample” and an “inventive antibody” into contact to thereby form a    complex of “the peptide consisting of the amino acid sequence    represented by SEQ ID NO:1 immobilized on the solid phase—the    inventive antibody” and a complex of “the peptide comprising the    amino acid sequence represented by SEQ ID NO:1 in the sample—the    inventive antibody”;-   (b) detecting at least one of the complex of “the peptide consisting    of the amino acid sequence represented by SEQ ID NO:1 immobilized on    the solid phase—the inventive antibody” and the complex of “the    peptide comprising the amino acid sequence represented by SEQ ID    NO:1 in the sample—the inventive antibody”.

In step (a), the three members, namely, the “inventive antibody”, the“sample” and the “solid phase on which a peptide consisting of the aminoacid sequence represented by SEQ ID NO:1 is immobilized” may be broughtinto contact simultaneously, or, alternatively, the former two are firstbrought into contact and then into contact with the latter one, or thelatter two are first brought into contact and then into contact with theformer one. It is preferred that the former two are first brought intocontact and then into contact with the latter one.

Also, in step (b), among the complex of the “peptide consisting of theamino acid sequence represented by SEQ ID NO:1 immobilized on the solidphase—the inventive antibody” and the complex of the “peptide comprisingthe amino acid sequence represented by SEQ ID NO:1 in the sample—theinventive antibody”, only the former can be detected, or only the lattercan be detected, or both can be detected. In this inventive method, itis preferred to detect only the former. That is, the inventive method ispreferably carried out by steps comprising the following steps (a) to(c):

-   (a) bringing an “inventive antibody” into contact with a sample to    thereby form a complex-A of “the peptide comprising the amino acid    sequence represented by SEQ ID NO:1—the inventive antibody”;-   (b) bringing a “mixture comprising the complex-A and the inventive    antibody which does not form the complex-A” obtained in step (a)    into contact with the “solid phase on which a peptide consisting of    the amino acid sequence represented by SEQ ID NO:1 is immobilized”    to thereby form a complex of “the peptide consisting of the amino    acid sequence represented by SEQ ID NO:1 immobilized on the solid    phase—the inventive antibody”; and-   (c) detecting the complex formed in step (b).

Each step is described below in detail.

Step (a):

Step (a) is a step for bringing an “inventive antibody” into contactwith a sample to thereby form a complex-A of “the peptide comprising theamino acid sequence represented by SEQ ID NO:1—the inventive antibody”.

The explanation of the “sample” is similar to that described above.Also, the explanation of the “inventive antibody” is similar to thatdescribed above. The method for bringing the sample into contact withthe inventive antibody is not particularly limited, so long as theinventive antibody is brought into contact with the “peptide comprisingthe amino acid sequence represented by SEQ ID NO:1” in the sample.

The sample is brought into contact with the inventive antibody tothereby form a complex-A of “the peptide comprising the amino acidsequence represented by SEQ ID NO:1—the inventive antibody”. As aresult, a “mixture comprising the complex-A and the inventive antibodywhich does not form the complex-A” is obtained by step (a).

Step (b):

Step (b) is a step for bringing the “mixture comprising the complex-Aand the inventive antibody which does not form the complex-A” obtainedin step (a) into contact with a “solid phase on which a peptideconsisting of the amino acid sequence represented by SEQ ID NO:1 isimmobilized” to thereby form a complex of “the peptide consisting of theamino acid sequence represented by SEQ ID NO:1 immobilized on the solidphase—the inventive antibody”.

The explanation of the solid phase to which the “peptide consisting ofthe amino acid sequence represented by SEQ ID NO:1” is immobilized issimilar to that in Inventive method-1.

The explanation of the “peptide consisting of the amino acid sequencerepresented by SEQ ID NO:1” immobilized on the solid phase is alsosimilar to that described above. Instead of this peptide, a “peptideconsisting of the amino acid sequence represented by SEQ ID NO:2” or a“peptide consisting of the amino acid sequence represented by SEQ IDNO:3” can also be used.

Also, the explanation of the method for immobilizing the “peptideconsisting of the amino acid sequence represented by SEQ ID NO: I” onthe solid phase is similar to that in Inventive method-1, and may use ausual method such as a physical adsorption method and a covalent bondmethod. Among these, a physical adsorption method is preferred becauseof its simple operation and wide use.

The contact between the “solid phase on which a peptide consisting ofthe amino acid sequence represented by SEQ ID NO:1 is immobilized” andthe mixture obtained in step (a) can be carried out as described above.Similarly to the above, the solid phase and the liquid phase areseparated after the reaction, and it is preferred that the surface ofthe solid phase is washed with a washing solution, if necessary, toremove any non-specifically adsorbed substances or non-reactedcomponents in the sample. The washing solution which can be used is alsosimilar to that described above.

As a result of bringing the “solid phase on which the peptide consistingof the amino acid sequence represented by SEQ ID NO:1 is immobilized”into contact with the mixture obtained in step (a), the “inventiveantibody which does not form the complex-A” is bound to the “peptideconsisting of the amino acid sequence represented by SEQ ID NO: I” tothereby form a complex of “the peptide consisting of the amino acidsequence represented by SEQ ID NO:1 immobilized on the solid phase—theinventive antibody”.

Step (c):

Step (c) is a step for detecting the complex formed in step (b). Themethod for detecting the complex is not particularly limited. When the“inventive antibody” which is labeled with a label or is capable ofbeing labeled with a label is used, the label is detected by a methodsimilar to that in Inventive method-1 to thereby detect the complex.Furthermore, the detection of the complex is preferably carried out byusing an “antibody which specifically binds to the inventive antibodyand which is labeled with a label or is capable of being labeled with alabel”.

Also, the explanations of the “antibody which specifically binds to theinventive antibody”, the label which can be used for labeling, alabeling method, a label detecting method and the like are similar tothose in Inventive method-1. However, since this method is an inhibitionmethod, detection of the label in a large amount means that theinventive antibody which does not form the complex-A of “the peptidecomprising the amino acid sequence represented by SEQ ID NO:1—theinventive antibody” is present in a large amount (correspondingly, thecomplex-A is present in a small amount), that is, the “peptidecomprising the amino acid sequence represented by SEQ ID NO:1” ispresent in a small amount in the sample.

<3>-1 Inventive Kit 1

Inventive kit-1 is a kit for assaying a “peptide comprising the aminoacid sequence represented by SEQ ID NO:1”, which comprises the followingcomponents (A) and (B):

-   (A) a solid phase; and-   (B) an inventive antibody.

The inventive antibody used in Inventive kit-1 is preferably one whichis labeled with a label or is capable of being labeled with a label.

The explanations of the “solid phase”, the “inventive antibody”, the“label”, the method for labeling the antibody with a label and the“peptide comprising the amino acid sequence represented by SEQ ID NO:1”as an assaying target in Inventive kit-1 are similar to those in“<2>-Inventive method”. This inventive kit can be used for assaying the“peptide comprising the amino acid sequence represented by SEQ ID NO:1”by means of direct ELISA.

<3>-2 Inventive Kit 2

Inventive kit-2 is a kit for assaying a “peptide comprising the aminoacid sequence represented by SEQ ID NO:1”, which comprises the followingcomponents (A) and (13):

-   (A) a solid phase on which an inventive antibody (primary antibody)    is immobilized; and-   (B) an inventive antibody (secondary antibody).

The “secondary antibody” used in Inventive kit-2 is preferably one whichis labeled with a label or is capable of being labeled with a label.

The explanations of the “inventive antibodies (primary antibody,secondary antibody)”, the “solid phase on which an inventive antibody(primary antibody) is immobilized”, the “label”, the method for labelingthe antibody with a label, and the “peptide comprising the amino acidsequence represented by SEQ ID NO:1” as an assaying target in Inventivekit-2 are similar to those in “<2>-Inventive method”. This inventive kitcan be used for assaying the “peptide comprising the amino acid sequencerepresented by SEQ ID NO:1” by means of a sandwich method.

<3>-3 Inventive Kit 3

Inventive kit-3 is a kit for assaying a “peptide comprising the aminoacid sequence represented by SEQ ID NO:1”, which comprises the followingcomponents (A), (B) and (C):

-   (A) a solid phase on which a peptide consisting of the amino acid    sequence represented by SEQ ID NO:1 is immobilized;-   (B) an inventive antibody; and-   (C) an antibody which specifically binds to the inventive antibody    and which is labeled with a label or is capable of being labeled    with a label.

The explanations of the “solid phase on which a peptide consisting ofthe amino acid sequence represented by SEQ ID NO:1 is immobilized”, the“inventive antibody”, the “antibody which specifically binds to theinventive antibody”, the “label”, the method for labeling the antibodywith a label and the “peptide comprising the amino acid sequencerepresented by SEQ ID NO:1” as an assaying target in Inventive kit-3 aresimilar to those in “<2>-Inventive method”. This inventive kit can beused for assaying a “peptide comprising the amino acid sequencerepresented by SEQ ID NO:1” by means of an inhibition method.

Assay using Inventive kit-1 can be carried out according to Inventivemethod-1, assay using Inventive kit-2 can be carried out according toInventive method-2, and assay using Inventive kit-3 can be carried outaccording to Inventive method-3.

<4> Inventive Detection Method

The inventive detection method is a method for detecting a bacterialpneumonia wherein an antigen in a sample which can be detected by an“inventive antibody” or an “antibody capable of specifically binding toCAP18” is assayed and, as a result, a bacterial pneumonia in a patientfrom which the sample is obtained is detected.

The “inventive antibody” and the “antibody capable of specificallybinding to CAP18” is preferably one which is labeled with a label or iscapable of being labeled with a label.

The explanations of the “inventive antibody”, the “label”, the methodfor labeling the antibody with a label, the “sample” and the “peptidecomprising the amino acid sequence represented by SEQ ID NO:1” as anassaying target in this inventive detection method are similar to thosein “<2>-Inventive method”.

CAP18 is preferably mammalian CAP18, and more preferably human CAP18.

The “antibody capable of specifically binding to CAP18” is notparticularly limited, so long as it can specifically bind to CAP18, andthe binding site is not particularly limited.

The inventive detection method is preferably carried out immunologicallyby using the “inventive antibody” or the “antibody capable ofspecifically binding to CAP18”, and examples include “<2>-Inventivemethod” described above. The “antibody capable of specifically bindingto CAP18” can be used, instead of the “inventive antibody”.

In the inventive detection method, the detection of a bacterialpneumonia is preferably carried out by evaluating or monitoring thepresence or absence of infection, degree or type of the bacterialpneumonia. Specifically, these detections can be carried out bycomparing the assaying results of a sample containing an antigen capableof being detected by the “inventive antibody” or the “antibody capableof specifically binding to CAP18” with the assaying results of a samplewhich does not contain the antigen.

The detection of CAP18 is not limited to the above-described method, andmay be carried out by a usual peptide detection method, such as highperformance liquid chromatography

<5> Inventive Diagnostic Kit

The inventive diagnostic kit is a kit for diagnosing a bacterialpneumonia, which comprises an “inventive antibody”.

The “inventive antibody” used in the inventive diagnostic kit ispreferably one which is labeled with a label or is capable of beinglabeled with a label.

The explanations of the “inventive antibody”, the “label” and the methodfor labeling the antibody with a label in this inventive diagnostic kitis similar to those in “<2>-Inventive method” described above.

The inventive diagnostic kit is preferably a kit of either one ofinventive kits 1 to 3, and assay using an Inventive kit-1 can be carriedout according to Inventive method-1, assay using an Inventive kit-2 canbe carried out according to Inventive method-2, and assay using anInventive kit-3 can be carried out according to Inventive method-3.

<6> Inventive Diagnostic Agent

The inventive diagnostic agent is an agent for diagnosing a bacterialpneumonia, which comprises an “inventive antibody”.

The “inventive antibody” used in the inventive diagnostic agent ispreferably one which is labeled with a label or is capable of beinglabeled with a label.

The explanations of the “inventive antibody”, the “label” and the methodfor labeling the antibody with a label in this inventive diagnosticagent are similar to those in “<2>-Inventive method” described above.

The inventive diagnostic agent can contain a pharmaceutically acceptablecarrier.

The present invention is described in detail based on Examples.

EXAMPLE 1

Production of Inventive Antibody:

(1) Production of Monoclonal Antibody

A peptide consisting of the amino acid sequence represented by SEQ IDNO:1 (FR KSKEK IGKEF KRIVQ RIKDF LRNLV; hereinafter referred to as “27amino acids-peptide”) was synthesized, bound to a hemocyanin (keyholelimpet hemocyanin), and then intraperitoneally administered to a mouse(Balb/c) (purchased from Clea Japan, 8 week-old) 4 times in total at 25μg as an initial dose and thereafter at 10 μg for immunization (using asan adjuvant a Freund complete adjuvant (manufactured by Wako PureChemical) initially and thereafter a Freund incomplete adjuvant(manufactured by Wako Pure Chemical)). Spleen cells were taken from theimmunized mouse, and then subjected to a cell fusion with mouse myelomacells (cell line: P3-X63-Ag8.653 (653: ATCC No. CRL 1588)) usingpolyethylene glycol 4000 (PEG4000) to thereby prepare a hybridoma.

The resultant hybridoma was proliferated continuously, and a hybridomacell line which continuously produces an antibody which specificallybinds to the 27 amino acids-peptide was selected.

The selected hybridoma cell line was cultured in a hollow fiber cellculture device using a serum-free medium (trade name: CD hybridoma,manufactured by Invitrogen), and the culture supernatant was taken anddialyzed against PBS to obtain a monoclonal antibody (Toyo6E3). Theantibody belongs to the subclass IgG1.

(2) Production of Polyclonal Antibody

A gene encoding the 27 amino acids-peptide was cloned into pET17b vector(pET17b-CAP18), then a region encoding the sequence excluding the signalpeptides (420 bp of 91-510, 140 amino acids) was then further insertedinto pET19b-CAP18 for re-cloning, and then transformation was carriedout in E. coli DH5α. The inserted gene sequence was confirmed and thentransformed again in E. coli BL21 (DE3) for protein expression, whichwas induced by IPTG (isopropyl-β-D-thiogalactopyranoside) to express theprotein. The protein was subjected to affinity purification using anNi-NTA (manufactured by Novagene) to obtain recombinant CAP18.

The human-derived recombinant CAP18 (rCAP18) was subcutaneouslyadministered to a rabbit at 0.1 μg for immunization (by using as anadjuvant a Freund complete adjuvant (manufactured by Wako Pure Chemical)initially and thereafter a Freund incomplete adjuvant (manufactured byWako Pure Chemical)). Two to three weeks after the initial immunization,booster immunization (0.1 to 0.2 mg/rabbit, 4 to 6 times) was carriedout by a standard method, and about 1 week after the final immunization,the blood was taken and the serum was separated. The serum was heated toinactivate complements, and then subjected to 33% saturated ammoniumsulfate precipitation to thereby obtain a polyclonal antibody.

EXAMPLE 2

Immunostaining of Cell by Monoclonal Antibody Toyo6E3:

Using the monoclonal antibody (Toyo6E3) produced in Example 1(1) as aprimary antibody and a goat anti-mouse IgG antibody (manufactured byJackson) labeled with an Alexa Fluor® 488 (manufactured by MolecularProbes) as a secondary antibody, human peripheral neutrophiles werestained according to a usual method. The fluoromicroscopic image(magnification: 1000) is shown in FIG. 1A.

The immune electromicroscopic images of human peripheral neutrophilesusing Toyo6E3 as a primary antibody and a goat anti-mouse IgG antibody(manufactured by Jackson) labeled with a 20 nm gold particle(manufactured by British Bioicell) as a secondary antibody are shown inFIGS. 1B (magnification: 5000) and 1C (magnification: 10000).

FIG. 1 shows that the monoclonal antibody Toyo6E3 can be used in cellstaining.

Also, FIG. 1A shows that a region which was considered to be thecytoplasm of the neutrophile can be stained by Toyo6E3. Furthermore,FIGS. 1B and C show that granulocytes of the neutrophile can be stainedby Toyo6E3.

EXAMPLE 3

Detection and Determination of Peptide by Direct ELISA:

The 27 amino acids-peptide and a “peptide consisting of the amino acidsequence represented by SEQ ID NO:2” (KEF KRIVQ RIKDF LRNLV; hereinafterreferred to as “18 amino acids-peptide”) were each dissolved in acarbonate buffer (pH 9.6) at various concentrations, and these solutionswere added to wells of a polystyrene microtiter plate. Thereafter, theplate was stored at about 22° C. for 1 hour to physically adsorb thepeptide to the plate. Subsequently, the solution was removed and thesurface of the plate was washed with a phosphate buffer (pH 7.5)supplemented with 0.05% Tween 20. Each peptide immobilized on this platewas assayed by the method (i) or (ii) described below.

-   (i) A method in which, after reacting the monoclonal antibody    produced in Example 1(1) (Toyo6E3) as a primary antibody and a goat    anti-mouse IgG antibody (manufactured by Jackson) labeled with a    horse radish peroxidase (HRP) as a secondary antibody, a TMB    chromogenic substrate (manufactured by Nippon Bio-Rad) was used to    develop a color, and an absorption at 450 nm was measured to thereby    assay the peptide immobilized on the solid phase.-   (ii) A method in which, after reacting the polyclonal antibody    produced in Example 1(2) as a primary antibody and a goat    anti-rabbit IgG antibody labeled with the HRP as a secondary    antibody, a TMB chromogenic substrate was used to develop a color,    and an absorption at 450 nm (OD450) was measured to thereby assay    the peptide immobilized on the solid phase.

The results of method (i) are shown in FIG. 2A, and the results ofmethod (ii) are shown in FIG. 2B. The curve indicated by “B” in thedrawings corresponds to the plate on which the 27 amino acids-peptidewas immobilized, and the curve indicated by “J” in the drawingscorresponds to the plate on which the 18 amino acids-peptide wasimmobilized.

FIG. 2A shows that both of the 27 amino acids-peptide and the 18 aminoacids-peptide can be detected and determined by the direct ELISA usingthe “monoclonal antibody Toyo6E3”. Also, since Toyo6E3 shows areactivity to the 18 amino acids-peptide at a degree similar to that tothe 27 amino acids-peptide, it was suggested that it specifically reactswith the 18 amino acids-peptide.

Also, FIG. 2B shows that both of the 27 amino acids-peptide and the 18amino acids-peptide can be detected and determined by the direct ELISAusing the “polyclonal antibody produced in Example 1(2)”. However,because of its low reactivity to the 18 amino acids-peptide, thispolyclonal antibody was indicated to be suitable for the detection anddetermination of the 27 amino acids-peptide.

Furthermore, these results suggest that this polyclonal antibody mainlyrecognizes the region which is contained in the 27 amino acids-peptidebut not contained in the 18 amino acids-peptide (SEQ ID NO:3;FRKSKEKIG).

EXAMPLE 4

Detection and Determination of CAP18 by Indirect ELISA (SandwichMethod):

The polyclonal antibody produced in Example 1(2) was dissolved in aphosphate buffer (pH 7.4), and the solution was added to wells of apolystyrene microtiter plate. Thereafter, the plate was stored at about22° C. for 1 hour to physically adsorb the peptide to the plate.Subsequently, the surface of the plate was washed with a phosphatebuffer (pH 7.4) supplemented with 0.05% Tween 20, and then blocked witha phosphate buffer supplemented with 1% BSA (bovine serum albumin). Tothis plate, the peptide and recombinant CAP18 (rCAP18; derived fromhuman) were added at various concentrations, followed by incubation at22° C. for 3 hours. After the incubation, the plate was washed with aphosphate buffer.

Then, the monoclonal antibody (Toyo6E3) produced in Example 1(1)(secondary antibody) was added to the plate, followed by incubation at22° C. for 2 hours. After the incubation, the plate was washed with aphosphate buffer.

Then, a goat anti-mouse IgG antibody labeled with the HRP was allowed toreact similarly, a TMB chromogenic substrate was used to develop acolor, and an absorption at 450 nm (OD450) was measured to therebydetermine the peptide or rCAP18. The results are shown in FIG. 3.

FIG. 3 shows that CAP18 can also be detected and determined by theindirect ELISA (sandwich method) using the “monoclonal antibody Toyo6E3”or the “polyclonal antibody produced in Example 1(2)”.

EXAMPLE 5

Detection of CAP18 in Cell Extract by western blotting:

Proteins in cell extracts of human peripheral blood mononuclear cells(monocytes), human peripheral neutrophiles (PMN), a humanmonocyte-derived cell line (THP-1) and a mouse macrophage-like cell line(J774.1) were separated by SDS-polyacrylamide gel electrophoresis, andelectrically transferred onto a nitrocellulose membrane. This membranewas blocked with a 3% skimmed milk, and then allowed to react with themonoclonal antibody (Toyo6E3) produced in Example 1(1). Then, the goatanti-mouse IgG antibody labeled with HRP was added as a secondaryantibody, and allowed to react similarly, and then the band developed byECL was detected. The results are shown in FIG. 4.

FIG. 4 shows that CAP18 can also be detected and determined by Westernblotting using the “monoclonal antibody Toyo6E3”.

It was also suggested that, in the human peripheral neutrophilefraction, 18 kDa-size CAP18 is present in a large amount and a lowermolecular antibody-binding protein (considered to be formed by enzymaticcleavage of the lipopolysaccharide-binding region (LPS-binding region)of the CAP18 molecule) is also present in a small amount. A small amountof the 18 kDa CAP18 was also detected in the human peripheral monocytefraction, whereas no substance which binds to Toyo6E3 was detected inthe human monocyte-derived THP-1 or the mouse macrophage-like cell lineJ774.1.

EXAMPLE 6

Detection of CAP18 in Serum by Immunoprecipitation:

Using the monoclonal antibody produced in Example 1(1) (Toyo6E3), CAP18contained in the serum of healthy persons (n=5) was immunoprecipitated.The immunoprecipitated material was detected by Western blotting usingthe same monoclonal antibody. The results are shown in FIG. 5. In thisconnection, the procedure of Western blotting was similar to that inExample 5.

FIG. 5 shows that the “monoclonal antibody Toyo6E3” can be also used inthe immunoprecipitation and that CAP18 can be detected by theimmunoprecipitation using this antibody.

EXAMPLE 7

Release of CAP18 from Human Neutrophiles Stimulated by fMLP:

Human peripheral neutrophiles (PN) or mononuclear cells (monocytes)(2×10⁶/well/200 ml) were incubated in the presence of 0 nM, 1 nM or 10nM formylmethionylleucylphenylalanine (fMLP) at 37° C. for 90 minutes.Thereafter, each of a culture supernatant fraction (150 ml) and acell-rich fraction (50 ml) was recovered, and in the latter fraction,cells were lysed to prepare an extract.

The amount of CAP18 contained in each of the supernatant fractions andcell extracts was assayed by the indirect ELISA using the monoclonalantibody (Toyo6E3) produced in Example 1(1). The results are shown inFIG. 6B. Also, based on the data in FIG. 6B, the “amount of the releasedCAP18” and the “amount of the intracellular CAP18” were estimated andthe results are shown in FIG. 6A. The fraction recovered in FIG. 6B wasalso detected by Western blotting using the monoclonal antibody(Toyo6E3), and the results are shown in FIG. 6C. The procedure ofWestern blotting was similar to that in Example 5, and the cell-richfraction was used after 3-fold dilution.

It was found from FIG. 6C that CAP18 is released into a culturesupernatant in response to the fMLP stimulation and that the releasedepends on the fMLP stimulation (concentration) (FIGS. 6A and B), by theELISA using the monoclonal antibody Toyo6E3. The size of the releasedCAP18 was 18 kDa, and no fragment of a small molecular weights wasdetected (FIG. 6C).

EXAMPLE 8

The indirect ELISA (sandwich method) described in Example 4 was used toassay CAP18 in sputum of each of serious pneumonia patients havingdifferent causative bacteria. The results are shown below. Assayed valueSubject to be assayed of CAP18 Patient A (disease: Klebsiela pneumonia)1.486 μg/ml Patient B (disease: methicillin-resistant 1.682 μg/mlStaphylococcus aureus)

In any patients, the CAP18 concentration in the sputum was markedlyincreased in comparison with comparative examples (control) shown below.

As comparative examples, the CAP18 concentration present in sputum ofeach of patients who exhibited no particular inflammatory sings and werebedridden for a long period and in sputum of a healthy human wereassayed by the indirect ELISA (sandwich method) as described above. Theresults are shown below. Assayed value Subject to be assayed of CAP18Patient C (Pseudomonas aeruginosa colonization, Not detected No fever,No increase of leukocytes) Patient D (Pseudomonas aeruginosa andStaphylococcus 0.674 μg/ml colonization, No fever, No increasedleukocyte) Healthy person E (smoker, healthy) Sample 1 Not detectedSample 2 Not detected

In any patients of the comparative examples, CAP18 was not detected atall or was detected only at a slightly increased level. No CAP18 wasdetected in the healthy person.

Based on the results described above, CAP18 in sputum can serve as anindex which gives a marked reflection of human neutrophile-derivedalveolar and interstitial inflammatory signs, and early diagnosis andmonitoring of a bacterial pneumonia can be carried out by assaying CAP18in a sample such as sputum.

EXAMPLE 9

Production of Inventive Kit:

-   (1) Inventive kit-1 having the following constitution was produced.-   1. 96-Well immunoplate 1-   2. Monoclonal antibody (Toyo6E3) produced in Example 1(1) 1 (primary    antibody)-   3. HRP-Labeled goat anti-mouse IgG antibody 1 (secondary antibody)-   4. TMB Solution 1-   5. Aqueous hydrogen peroxide 1-   6. Reaction-stopping solution (1M HCl) 1-   (2) Inventive kit-2 having the following constitution was produced.-   1. 96-Well immunoplate on which the polyclonal antibody 1 (Toyo6E3)    produced in Example 1(2) is immobilized-   2. Monoclonal antibody (Toyo6E3) produced in Example 1(1) 1 (primary    antibody)-   3. HRP-Labeled goat anti-mouse IgG antibody 1 (secondary antibody)-   4. TMB Solution 1-   5. Aqueous hydrogen peroxide 1-   6. Reaction-stopping solution (1M HCl) 1-   (3) Inventive kit-2 having the following constitution was produced.-   1. 96-Well immunoplate on which the 27 amino acids 1-peptide is    immobilized-   2. Monoclonal antibody (Toyo6E3) produced in Example 1(1) 1-   3. HRP-Labeled goat anti-mouse IgG antibody 1 (secondary antibody)-   4. TMB Solution 1-   5. Aqueous hydrogen peroxide 1-   6. Reaction-stopping solution (1M HCl) 1

While the present invention has been described in detail and withreference to specific embodiments thereof, it will be apparent to one ofskill in the art that various changes and modifications can be madetherein without departing from the spirit and scope thereof.

This application is based on Japanese application Nos. 2002-2213040 and2003-70932 filed on Jul. 22, 2002 and Mar. 14, 2003, respectively, theentire contents of which are incorporated hereinto by reference. Allreferences cited herein are incorporated in their entirety.

INDUSTRIAL APPLICABILITY

The inventive antibody is an antibody which specifically binds to apeptide characteristic of CAP18, and is very useful because it can beused as a tool for detecting or assaying CAP18. The inventive antibodyis highly homogeneous and reproducible, and can also permanently beproduced in a large amount, resulting advantageously in remarkablereduction in production costs. Furthermore, the inventive antibody isvery useful since it can be used in the inventive method or inproduction of the inventive kit.

The inventive method is extremely useful since it can be applied to aresearch reagent or a diagnostic agent for diseases relating to CAP18and the like and can also be possibly to applied to monitoring of thediseases and the like. For example, it can be used in a method forevaluating respiratory diseases such as chronic pulmonary disease,chronic airway disease, acute pulmonary disease, inflammatory pulmonarydisease, adult respiratory distress syndrome (ARDS), bacterialpneumonia, interstitial pneumonia and upper airway bronchitis, whichcomprises assaying a CAP18 amount in a living sample and relating theassayed result to the diseases.

Furthermore, the inventive kit is extremely useful since the inventivemethod can carried out more rapidly and more conveniently by using theinventive kit.

Free Text of Sequence Listing

-   SEQ ID NO:1—explanation of artificial sequence: synthetic peptide-   SEQ ID NO:2—explanation of artificial sequence: synthetic peptide-   SEQ ID NO:3—explanation of artificial sequence: synthetic peptide

1. An antibody which binds to a “peptide consisting of the amino acidsequence represented by SEQ ID NO:1”.
 2. The antibody according to claim1, which specifically binds to a “peptide consisting of the amino acidsequence represented by SEQ ID NO:2”.
 3. The antibody according to claim1, which is a monoclonal antibody.
 4. The antibody according to claim 1,which belongs to the immunoglobulin subclass IgG1.
 5. The antibodyaccording to claim 1, which specifically binds to a “peptide consistingof the amino acid sequence represented by SEQ ID NO:3”.
 6. The antibodyaccording to claim 1, which is a polyclonal antibody.
 7. A method forassaying a “peptide comprising the amino acid sequence represented bySEQ ID NO:1” in a sample, which comprises using an antibody described inof claim
 1. 8. The method according to claim 7, wherein said assaying ofthe “peptide comprising the amino acid sequence represented by SEQ IDNO:1” is carried out by steps comprising the following steps (a) and(b): (a) bringing a solid phase into contact with a sample to therebyimmobilize the “peptide comprising the amino acid sequence representedby SEQ ID NO:1” in the sample on the solid phase; and (b) detecting the“peptide comprising the amino acid sequence represented by SEQ ID NO:1”immobilized on the solid phase in step (a) by using an “antibodydescribed in claim 1”.
 9. The method according to claim 8, wherein the“antibody described in claim 1” is labeled with a label or is capable ofbeing labeled with a label.
 10. The method according to claim 8, whereinsaid detecting of the “peptide comprising the amino acid sequencerepresented by SEQ ID NO:1” immobilized on the solid phase is carriedout by further using an “antibody which specifically binds to theantibody described in of claim 1 and which is labeled with a label or iscapable of being labeled with a label”.
 11. The method according toclaim 7, wherein said assaying of the “peptide comprising the amino acidsequence represented by SEQ ID NO:1” is carried out by steps comprisingthe following steps (a) and (b): (a) bringing a “solid phase on which anantibody described in claim 1 as a primary antibody is immobilized”, a“sample”, and an “antibody described in claim 1 as a secondary antibody”into contact to thereby form a sandwich complex of “the primary antibodyimmobilized on the solid phase—the peptide comprising the amino acidsequence represented by SEQ ID NO:1—the secondary antibody”; and (b)detecting the sandwich complex formed in step (a).
 12. The methodaccording to claim 11, wherein said assaying of the “peptide comprisingthe amino acid sequence represented by SEQ ID NO:1” is carried out bysteps comprising the following steps (a) to (c): (a) bringing a sampleinto contact with a “solid phase on which an antibody described in claim1 as a primary antibody is immobilized” to thereby form a complex of“the primary antibody immobilized on the solid phase—the peptidecomprising the amino acid sequence represented by SEQ ID NO:1”; (b)bringing an “antibody described in claim 1 as a secondary antibody” intocontact with the solid phase to thereby form a sandwich complex of “theprimary antibody immobilized on the solid phase—the peptide comprisingthe amino acid sequence represented by SEQ ID NO:1—the secondaryantibody”; and (c) detecting the sandwich complex formed in step (b).13. The method according to claim 11, wherein the secondary antibody islabeled with a label or is capable of being labeled with a label. 14.The method according to claim 11, wherein said detecting of the complexis carried out by using an “antibody which specifically binds to thesecondary antibody and which is labeled with a label or is capable ofbeing labeled with a label”.
 15. The method according to claim 7,wherein said assaying of the “peptide comprising the amino acid sequencerepresented by SEQ ID NO:1” is carried out by steps comprising thefollowing steps (a) and (b): (a) bringing a “solid phase on which apeptide consisting of the amino acid sequence represented by SEQ ID NO:1is immobilized”, a “sample”, and an “antibody described in claim 1” intocontact to thereby form a complex of “the peptide consisting of theamino acid sequence represented by SEQ ID NO:1 immobilized on the solidphase—the antibody described in claim 1” and a complex of “the peptidecomprising the amino acid sequence represented by SEQ ID NO:1 in thesample—the antibody described in claim 1”; and (b) detecting at leastone of the complex of “the peptide consisting of the amino acid sequencerepresented by SEQ ID NO:1 immobilized on the solid phase—the antibodydescribed in claim 1” and the complex of “the peptide comprising theamino acid sequence represented by SEQ ID NO:1 in the sample—theantibody according to claim 1”.
 16. The method according to claim 15,wherein said assaying of the “peptide comprising the amino acid sequencerepresented by SEQ ID NO:1” is carried out by steps comprising thefollowing steps (a) to (c): (a) bringing a sample into contact with an“antibody described in claim 1” to thereby form a complex-A of “thepeptide comprising the amino acid sequence represented by SEQ IDNO:1—the antibody described in claim 1”; (b) bringing a “mixturecomprising the complex-A and the antibody described in claim 1 whichdoes not form the complex-A” obtained in step (a) into contact with a“solid phase on which the peptide consisting of the amino acid sequencerepresented by SEQ ID NO:1 is immobilized” to thereby form a complex of“the peptide consisting of the amino acid sequence represented by SEQ IDNO:1 immobilized on the solid phase—the antibody described in claim 1”;and (c) detecting the complex formed in step (b).
 17. The methodaccording to claim 15, wherein the “antibody described in claim 1” islabeled with a label or is capable of being labeled with a label. 18.The method according to claim 15, wherein said detecting of the complexis carried out by using an “antibody which specifically binds to theantibody according to claim 1 and which is labeled with a label or iscapable of being labeled with a label”.
 19. The method according toclaim 7, wherein the sample is a body fluid.
 20. A kit for assaying a“peptide comprising the amino acid sequence represented by SEQ ID NO:1”,which comprises the following components (A) and (B): (A) a solid phase;and (B) an antibody described in claim
 1. 21. The kit according to claim20, wherein the “antibody described in claim 1” is labeled with a labelor is capable of being labeled with a label.
 22. A kit for assaying a“peptide comprising the amino acid sequence represented by SEQ ID NO:1”,which comprises the following components (A) and (B): (A) a solid phaseon which an antibody described in claim 1 as a primary antibody isimmobilized; and (B) an antibody described in claim 1 as a secondaryantibody.
 23. The kit according to claim 22, wherein the secondaryantibody is labeled with a label or is capable of being labeled with alabel.
 24. A kit for assaying a “peptide comprising the amino acidsequence represented by SEQ ID NO:1”, which comprises the followingcomponents (A), (B) and (C): (A) a solid phase on which a peptideconsisting of the amino acid sequence represented by SEQ ID NO:1 isimmobilized; (B) an antibody described in claim 1; and (C) an antibodywhich specifically binds to the antibody described in claim 1 and whichis labeled with a label or is capable of being labeled with a label. 25.A method for detecting a bacterial pneumonia, which comprises assayingan antigen in a sample which can be detected by an “antibody describedin claim 1” or an “antibody capable of specifically binding to CAP18” tothereby detect a bacterial pneumonia in a patient from which the sampleis obtained.
 26. The method according to claim 25, wherein the antigenin the sample is selected from the group consisting of a “peptidecomprising the amino acid sequence represented by SEQ ID NO:1”, a“peptide comprising the amino acid sequence represented by SEQ ID NO:2”,a “peptide comprising the amino acid sequence represented by SEQ IDNO:3”, and CAP18.
 27. The method according claim 25, wherein saidassaying is immunologically carried out by using an antibody selectedfrom the group consisting of an “antibody capable of binding to apeptide consisting of the amino acid sequence represented by SEQ IDNO:1”, an “antibody capable of specifically binding to a peptideconsisting of the amino acid sequence represented by SEQ ID NO:2”, an“antibody capable of specifically binding to a peptide consisting of theamino acid sequence represented by SEQ ID NO:3”, and an “antibodycapable of specifically binding to CAP18”.
 28. The method according toclaim 25, wherein said detecting of a bacterial pneumonia is carried outby evaluating or monitoring the presence or absence of infection, degreeor type of the bacterial pneumonia.
 29. The method according to claim25, wherein said assaying is carried out by a method described in claim7.
 30. A kit for diagnosing a bacterial pneumonia, which comprises anantibody described in claim
 1. 31. The kit according to claim 30, whichconsists of any one of a kit described in claim
 20. 32. A diagnosticagent which comprises, as an active ingredient, an antibody according toclaim 1.